A study of resuscitation-promoting factors in Mycobacterium tuberculosis.

Tuberculosis is a major threat to human health. About one third of the world's population is latently infected with Mycobacterium tuberculosis . In these cases the bacillus is in a state of low metabolic activity, making eradication difficult with conventional chemotherapy, which targets actively metabolizing organisms. The mechanisms by which M. tuberculosis reactivates to cause disease are currently unknown but a better understanding could greatly improve the treatment of tuberculosis. Resuscitation-promoting factor is a protein first identified in the supernatant of stationary phase cultures of Micrococcus luteus. It is active in picomolar concentrations, increasing the number of culturable M. luteus cells from dormant populations and shortening the lag phase of growth of small inocula. Bioinformatic searches reveal over 40 examples of rpf-ke genes in the high G-C cohort of Gram-positive bacteria, including M. tuberculosis , which contains five rpf gene orthologues. The work presented here investigated aspects of the M. tuberculosis Rpfs. Improvements in solubility of recombinant mycobacterial (M. tuberculosis and M. smegmatis) Rpfs were achieved by manipulating induction times and temperatures during protein expression and by using new hosts and vectors and producing novel fusion proteins. New assays were devised to measure the biological activity of recombinant Rpfs, using ATP bioluminescence of M. luteus cultures. A phage display library for M. tuberculosis was constructed, in an attempt to identify a protein receptor for Rpf. Rpf expression in human infection was investigated for the first time, using immunocytochemistry. Anti-Rpf antibodies were applied to human tissue sections infected with M. tuberculosis. Rpf was found to be located within epithelioid giant cells and in the immediate vicinity of acid-fast bacilli in necrotic centres. The presence of Rpf in human tuberculosis infection demonstrated in this work suggests that Rpfs may have a role in controlling dormancy of the bacilli in human disease